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Targeting proteins that are overexpressed in cancer cells is the major strategy of molecular imaging and drug delivery systems. The 67-kDa laminin receptor (67LR), also known as oncofetal antigen, is overexpressed in several types of cancer, including melanoma, multiple myeloma, cervical cancer and bile duct carcinoma. 67LR is involved in tumour growth, tumour metastasis and drug resistance. Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) directly binds to cell-surface 67LR and induces apoptosis through the protein kinase B (Akt)/endothelial nitric oxide synthase/nitric oxide/cyclic GMP (cGMP) axis. Here we report the optimum hydroxyl group for the utilization of EGCG as a novel fluorescent EGCG-mimic imaging probe based on 67LR agonist characters, including Akt activation and inhibitory effect on viable cell number in cancer cells. 67LR specific targeting is unambiguously confirmed with the use of a non-labelled EGCG competitive assay and 67LR knockdown. Importantly, this probe strongly binds to multiple myeloma cells compared with its binding to normal cells.

Anticancer agents can cause various side effects, including tissue damages/inflammatory reactions. Drug-responsive biomarkers are essential for evaluating drug toxicity in disease processes. S100 calcium-binding proteins A8/A9 (S100A8/A9) are highly expressed in neutrophils and monocytes/macrophages accumulated at inflammatory sites and are known to be related to tissue damage/inflammation; however, their response to drug toxicity has not been reported. Herein, we investigated the effects of anticancer agents (doxorubicin, cisplatin, and docetaxel) on S100A8/A9 gene expression profiles in four representative tissues (heart, kidney, liver, and lung) in normal C57BL/6J mice. Both S100A8/A9 expression was transiently or time-dependently elevated in four tissues within 48 h after dosing of the three anticancer agents under toxicity-inducing conditions. S100A8/A9 patterns differed among agents and tissues. This result suggests that S100A8/A9 is useful for evaluating anticancer agent-induced tissue damage. Metabolomic analysis revealed that some metabolites showed temporal patterns similar to that of S100A8/A9 expression. The amounts of fumarate (doxorubicin-treated heart), tyrosine (cisplatin-treated kidney), acetylcarnosine (doxorubicin-treated liver), and 2-phosphoglycerate (docetaxel-treated lung) showed similar patterns to that of S100A8/A9 expression. Although these metabolites showed different behaviors between tissues and serum, they may be useful marker candidates for evaluating anticancer agent-induced tissue damage at an earlier stage after dosing.

Epigallocatechin-3-O-gallate (EGCG)-induced cyclic guanosine monophosphate (cGMP) plays a crucial role in EGCG-induced cell death in various types of cancer cells. However, little is known regarding the early molecular events after cGMP induction. In this study, we showed that cGMP induction is sufficient to induce the phosphorylation of protein kinase C delta (PKCδ) at Ser664, the crucial kinase for EGCG-induced activation of acid sphingomyelinase (ASM). Using a chemical inhibitor library, we revealed that the inhibitors of the negative regulators of diacylglycerol strongly increase the effect of EGCG. We also showed that EGCG treatment increased phospholipase C (PLC) activity, and the same results were obtained with cGMP inducer treatment. EGCG-induced ASM activation was completely suppressed by pharmacological inhibition of PLC. Collectively, EGCG-induced cGMP activated the cGMP/PLC/PKCδ/ASM signaling axis in multiple myeloma cells.

Epigallocatechin 3-O-gallate (EGCG), the major catechin in green tea, exerts several beneficial effects in vivo. A previous study showed that cyclic guanosine monophosphate (cGMP) as a cellular second messenger has been shown to be an important mediator of the effects of EGCG. In general, long-term animal experiments are required to evaluate the functionality of phytochemicals such as EGCG. Thus, it might be beneficial to establish a marker for short-term evaluation of functional phytochemicals. In this study, the use of the plasma cGMP level was evaluated as a surrogate marker for the functional effectiveness of EGCG. Blood samples were collected from 6-wk-old C57BL/6J mice from 1 to 8 h after oral administration of EGCG (30 or 100 mg/kg). The upregulation of plasma cGMP levels elicited by the oral EGCG administration was evaluated. Additionally, it was confirmed that EGCG induced the expressions of intracellular Toll interacting protein (Tollip), which is mainly controlled by a cGMP signal in the spleen. These data indicated that the plasma cGMP levels could be a screening marker for the effect of EGCG.

Objective Metformin and glucagon like peptide-1 (GLP-1) prevent diabetic cardiovascular complications and atherosclerosis. However, the direct effects on hyperglycemia-induced oxidative stress in endothelial cells are not fully understood. Thus, we aimed to evaluate the effects of metformin and a GLP-1 analog, liraglutide on high glucose-induced oxidative stress. Methods Production of reactive oxygen species (ROS), activation of protein kinase C (PKC) and NAD(P)H oxidase, and changes in signaling molecules in response to high glucose exposure were evaluated in human aortic endothelial cells with and without treatment of metformin and liraglutide, alone or in combination. PKC-NAD(P)H oxidase pathway was assessed by translocation of GFP-fused PKCβ2 isoform and GFP-fused p47phox, a regulatory subunit of NAD(P)H oxidase, in addition to endogenous PKC phosphorylation and NAD(P)H oxidase activity. Results High glucose-induced ROS overproduction was blunted by metformin or liraglutide treatment, with a further decrease by a combination of these drugs. Exposure to high glucose caused PKCβ2 translocation and a time-dependent phosphorylation of endogenous PKC but failed to induce its translocation and phosphorylation in the cells treated with metformin and liraglutide. Furthermore, both drugs inhibited p47phox translocation and NAD(P)H oxidase activation, and prevented the high glucose-induced changes in intracellulalr diacylglycerol (DAG) level and phosphorylation of AMP-activated protein kinase (AMPK). A combination of these drugs further enhanced all of these effects. Conclusions Metformin and liraglutide ameliorate high glucose-induced oxidative stress by inhibiting PKC-NAD(P)H oxidase pathway. A combination of these two drugs provides augmented protective effects, suggesting the clinical usefulness in prevention of diabetic vascular complications.

Adipose triglyceride lipase (ATGL) was recently identified as a rate-limiting triglyceride (TG) lipase and its activity is stimulated by comparative gene identification-58 (CGI-58). Mutations in the ATGL or CGI-58 genes are associated with neutral lipid storage diseases characterized by the accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, is characterized by TG accumulation in coronary atherosclerotic lesions and in the myocardium. Recent reports showed that myocardial TG accumulation is significantly higher in patients with diabetes and is associated with impaired left ventricular diastolic function. Therefore, we investigated the roles of ATGL and CGI-58 in the development of myocardial steatosis in the diabetic state. Histological examination with oil red O staining showed marked lipid deposition in the hearts of diabetic fatty db/db mice. Cardiac triglyceride and diglyceride contents were greater in db/db mice than in db/+ control mice. Next, we determined the expression of genes and proteins that affect lipid metabolism, and found that ATGL and CGI-58 expression levels were decreased in the hearts of db/db mice. We also found increased expression of genes regulating triglyceride synthesis (sterol regulatory element-binding protein 1c, monoacylglycerol acyltransferases, and diacylglycerol acyltransferases) in db/db mice. Regarding key modulators of apoptosis, PKC activity, and oxidative stress, we found that Bcl-2 levels were lower and that phosphorylated PKC and 8-hydroxy-2'-deoxyguanosine levels were higher in db/db hearts. These results suggest that reduced ATGL and CGI-58 expression and increased TG synthesis may exacerbate myocardial steatosis and oxidative stress, thereby promoting cardiac apoptosis in diabetic mice.

We examined the effects of adipose triglyceride lipase (ATGL) on the initiation of atherosclerosis. ATGL was recently identified as a rate-limiting triglyceride (TG) lipase. Mutations in the human ATGL gene are associated with neutral lipid storage disease with myopathy, a rare genetic disease characterized by excessive accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, shows massive TG accumulation in both coronary atherosclerotic lesions and the myocardium. Recent reports show that myocardial triglyceride content is significantly higher in patients with prediabetes or diabetes and that ATGL expression is decreased in the obese insulin-resistant state. Therefore, we investigated the effect of decreased ATGL activity on the development of atherosclerosis using human aortic endothelial cells. We found that ATGL knockdown enhanced monocyte adhesion via increased expression of TNFα-induced intercellular adhesion molecule-1 (ICAM-1). Next, we determined the pathways (MAPK, PKC, or NFκB) involved in ICAM-1 up-regulation induced by ATGL knockdown. Both phosphorylation of PKC and degradation of IκBα were increased in ATGL knockdown human aortic endothelial cells. In addition, intracellular diacylglycerol levels and free fatty acid uptake via CD36 were significantly increased in these cells. Inhibition of the PKC pathway using calphostin C and GF109203X suppressed TNFα-induced ICAM-1 expression. In conclusion, we showed that ATGL knockdown increased monocyte adhesion to the endothelium through enhanced TNFα-induced ICAM-1 expression via activation of NFκB and PKC. These results suggest that reduced ATGL expression may influence the atherogenic process in neutral lipid storage diseases and in the insulin-resistant state.

Background Decreases in Sonic hedgehog ( SHH ) and CDX2 expression are associated with atrophy and intestinal metaplasia in the gastric mucosa. The pathogenesis of development of Barrett's oesophagus is still unclear. Objective To examine the gene expression of CDX2 and SHH and their signalling pathways in the columnar epithelium and the association with endoscopic appearance, gastric pH or bile acids. Subjects/Methods Sixty-three patients with metaplastic columnar epithelium of the lower oesophagus were studied. Whole biopsy specimens and microdissected tissues were examined for messenger RNA. Results BMP4 expression was significantly higher in patients with tubular mucosal patterns of columnar epithelium visualised by Narrow Band Imaging with magnification. The expression of SHH was significantly lower and that of CDX2 was higher in the goblet columnar epithelium than in non-goblet columnar epithelium. CDX2 expression was significantly higher in the patients with hypoacidity than in the others. BMP4 and PTCH1 expression was significantly higher in the group with higher concentrations of deoxycholic acid than in the group with lower concentrations. Conclusions SHH might be the initial factor inducing columnar metaplasia, and subsequent or simultaneous BMP4 stimuli might induce the CDX2 expression that causes goblet-cell metaplasia.

Recently, we have reported that (-)-epigallocatechin-3-O-gallate (EGCG) acts as an inhibitor of degranulation. However, the inhibitory mechanism for degranulation is still poorly understood. Here we show that suppression of exocytosis-related myosin II regulatory light chain phosphorylation and alteration of actin remodeling are involved in the inhibitory effect of EGCG on the calcium ionophore-induced degranulation from human basophilic KU812 cells. Surface plasmon resonance assay also revealed that EGCG binds to the cell surface, and the disruption of lipid rafts resulted in reduction of EGCG's ability. We have previously identified the raft-associated 67kDa laminin receptor (67LR) as an EGCG receptor on the cell surface. Treatment of the cells with anti-67LR antibody or RNA interference-mediated downregulation of 67LR expression abolished the effects of EGCG. These findings suggest that EGCG-induced inhibition of the degranulation includes the primary binding of EGCG to the cell surface 67LR and subsequent modulation of cytoskeleton.

(-)-Epigallocatechin-3-O-gallate (EGCG), a major green tea polyphenol, has previously exhibited a suppressive effect on the expression of the high-affinity IgE receptor (FcepsilonRI). This effect has been shown to be elicited by interaction with the plasma membrane microdomain lipid rafts. Recently, we have identified the 67 kDa laminin receptor (67LR) as a cell surface EGCG receptor that mediates an anti-cancer action. Here we show that the 67LR is highly associated with lipid rafts on human basophilic KU812 cells. Experiments using 67LR-enhanced and -reduced cells revealed that the EGCG's ability to downregulate FcepsilonRI expression correlated with the amount of 67LR. Thus, these results suggest that the lipid raft-associated 67LR plays an important role in mediating the FcepsilonRI-suppressive action of EGCG.

Meckel's diverticulum has been known to occur in 2% of the general population and is usually asymptomatic; it causes complications in 4% of cases. We have recently experienced a rare case of perforation of Meckel's diverticulum in a neonate delivered to a mother who had been treated with methimazole for hyperthyroidism during pregnancy. A 2-day-old neonate who was delivered by Cesarean section at 39 weeks' gestation underwent an emergency operation; he was diagnosed with gastrointestinal perforation, showing a large amount of air within the gastrointestinal tract, and pneumoperitoneum radio graphically. Laparotomy showed a perforated Meckel's diverticulum in the jejunum with a moderate amount of serous ascitic fluid. Segmental resection of the small bowel and intraperitoneal drainage were performed. A pathological examination for Meckel's diverticulum revealed a partially thinned muscular layer suggesting the existence of an increase in pressure within the diverticulum. The neonate needed to be administrated thyroid hormone for a hypothyroid state, which was possibly due to the maternal treatment with methimazole during pregnancy. We hypothesize that maternal treatment with methimazole during pregnancy may be associated with the formation of Meckel's diverticulum in a neonate; and it may be associated with an abdominal distention and subsequent perforation of Meckel's diverticulum with secondary hypothyroidism.

Background: Although duodenal adenomas are very common in familial adenomatous polyposis, we wished to emphasize our experience with finding nonpolypoid adenomas in the duodenum of patients with this disease. Methods: Duodenoscopy was performed in 23 patients with an established diagnosis of familial adenomatous polyposis or Gardner's syndrome. Results: Endoscopy revealed single or multiple nonpolypoid adenomas of the duodenum in 7 patients (30%). The lesions were smaller than 5 mm and were endoscopically recognized as flat or depressed reddish lesions; one lesion was completely flat and the remaining lesions were flat-topped elevations with a central depression. All of the lesions were histologically diagnosed as tubular adenoma with moderate epithelial atypia. Conclusion: These findings suggest that duodenal nonpolypoid adenomas are common in familial adenomatous polyposis or Gardner's syndrome and that careful surveillance endoscopy seems necessary in patients with this disease. (Gastrointest Endosc 1996;44:305-8.)

Background: Splicing and expression of alternative exons is a tissue specific feature that affects the cancer genome. Notably, pre-mRNA-splicing factor SRp20 plays an important role in regulating alternative splicing. Expression of CD44 and its splicing variants have been shown to regulate carcinogenesis and invasion. Herein, we investigated factors that regulate splicing of CD44 and expression of its variant CD44E in gastric cancer. We identified a novel role for dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), in regulating CD44 splicing and promoting gastric tumorigenesis. Methods:Pre-mRNA splicing was measured by utilizing a luciferase-based quantitative reporter system. Quantitative realtime RT-PCR and Western blot analysis were used to measure the mRNA and protein levels. Co-immunoprecipitation, invasion, and ubiquitination assays were performed to investigate the mechanism by which DARPP-32 regulates CD44 splicing . Results: Knockdown of endogenous DARPP-32 in MKN45 gastric cancer cells markedly reduced expression of CD44 V8-V10 (CD44E) splice variant. We found that overexpression of DARPP-32 in AGS gastric cancer cells, with low endogenous levels, significantly increased the splicing activity (P

G A A b st ra ct s Results: BEC-40 weeks and BEC-60 weeks had significantly higher proliferation rates compared to BEC-0 weeks and BEC-20 weeks (See Table 1). Although there was no statistically significant difference in proliferation rate between BEC-0 week and 20 week cells, the BEC60 week cells grew 2.75 fold higher compared to BEC-40 week cells. The apoptosis rate showed a reverse pattern with a 50 % suppression in BEC-60 week cells compared to BEC0 week cells. Serum-deprivation induced a 5.4-fold reduction in proliferation rate in BEC60 week cells compared to the BEC-40 week cells. Conclusion: The transformed BEC-60 week cells proliferate at a faster rate than BEC-0 weeks, 20 weeks, and 40 week cells. They also appear to be more resistant to apoptosis as a consequence of B4 exposure. However, proliferation of BEC-60 weeks is most affected by serum deprivation. Increased proliferation, reduced apoptosis, and serum dependence due to chronic B4 exposure in BEC cells are likely properties of dysplasia and neoplasia observed in BE progression to esophageal adenocarcinoma in vivo. We propose that this dynamic novel BEC model may be applied for future chemoprevention studies for BE-carcinogenesis. Table 1: Fold change between different phases of the BEC model in the presence and absence of serum in growth medium

Effect of Famotidine After Endoscopic Treatment on Bleeding Peptic Ulcers Tomoari Kamada, Jiro Hata, Hiroaki Kusunoki, Yoshinori Fujimura, Tatsuro Tanimoto, Soichiro Kido, Hiroshige Hamada, Ken Haruma Background and Aim: Peptic ulcers are the most common cause of upper gastrointestinal bleeding. The aim of our study was to investigate the effect of acid suppressive therapy after endoscopic treatment on bleeding peptic ulcer diseases. Patients and Methods: The 398 patients with bleeding peptic ulcers were divided into two groups those receiving drip infusion of PPI (40 mg/day) (n Z 165, 116 men, 49 women, mean age 59.8 yrs) and those receiving infusion of famotidine (40 mg/day) (n Z 233, 188 men, 45 women, mean age 61.5 yrs) after initial endoscopic treatment. The rate of recurrent bleeding, mortality, fasting duration, hospital stay, the rate and volume of blood transfusion were evaluated between the two groups in our study. Results: The rate of re-bleeding was 7.8% in the famotidine group and 7.2% in the PPI group, no significant differences were found. The mean hospital stay for the PPI group was significantly shorter than that for the famotidine group (PPI group 18.3 day vs famotidine group 21.4 day, p ! 0.05). Also, mortality, fasting duration, the rate and volume of blood transfusion were no statistically significant differences between the two groups. Conclusion: Our study demonstrated that the infusion of famotidine after endoscopic treatment is an effective drug for bleeding peptic ulcer diseases. M1347 Impact of Change in Upper Gastrointestinal Endoscope Findings in Japan Takashi Kawai, Kohei Kawakami, Kazuo Takei, Takashi Okamoto, Naomi Uemura Background: H. pylori infection rate in Japan has been reported to be high, but recently it is decreasing rapidly. H.pylori infection has also been associated with gastritis, gastric polyp, peptic ulcers, and gastric cancer. We examined the H. pylori infection rate and correlation of H.pylori infection with upper gastrointestinal endoscope (UGIE) finding. Method: Subject were 419 patients who received UGIE and examined serum IgG H.pylori antibody for health check from August 2003 to April 2004. Mean ages were 39.2 ,} 8.3 (22-58 age). We investigated UGIE findings reference to atrophic gastritis (mild, moderate, severe), reflex esophagitis (RE), gastric fundic polyp (FP), gastric hyperplastic polyp (HP), Gastric kammroetung (GK), nodular gastritis (NG), gastric ulcer (GU), duodenal ulcer (DU). Result: Total H. pylori infection rate was 33.7% (141/419). Infection rates were 15.7% in age 20-29, 28.0% in age 30-39, 34.3% in age 40-49 and 69.6% in age 50-59. Rates of mild, moderate, and severe of atrophic gastritis were 96.2%, 3.2% and 0% respectively in H. pylori-negative patients. On the other hand, rates of mild, moderate, and severe of atrophic gastritis were 26.2%, 50.4% and 23.4% respectively in H. pylori-positive patients. Rates of UGIE findings were 25.8% in RE, 39.9% in GK, 8.6% in FP, 0% in HP, 0% in NG, 1.1% in GU, 1.4% in DU in H.pylorinegative patients. Rates of UGIE findings were 16.3% in RE, 5.0% in GK, 0.7% in FP, 4.3% in HP, 4.3% in NG, 13.4% in GU, 21.9% in DU in H.pylori-positive patients. Conclusion: H. pylori infection rate decreased drastically compared to a report by Asaka et al 1) (infection rate were 25% in age 20-29, 43% in age 30-39, 78% in age 40-49). There were significant differences in UGIE findings between H. pylori-negative and H. pylori-positive. In the future it is possible that UGIE findings in Japan will resemble those in Western countries, because of expectation of more decrease in H. pylori infection rate in Japan. 1) Asaka M, Kimura T, Takeda H, et al. Gastroenterology 1992; 102: 760-766.